Departments of Radiology and Chemistry, Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, CA, 94305, USA.
Angew Chem Int Ed Engl. 2020 May 11;59(20):7864-7870. doi: 10.1002/anie.201916352. Epub 2020 Mar 20.
The pre-targeted imaging of enzyme activity has not been reported, likely owing to the lack of a mechanism to retain the injected substrate in the first step for subsequent labeling. Herein, we report the use of two bioorthogonal reactions-the condensation reaction of aromatic nitriles and aminothiols and the inverse-electron demand Diels-Alder reaction between tetrazine and trans-cyclooctene (TCO)-to develop a novel strategy for pre-targeted imaging of the activity of proteases. The substrate probe (TCO-C-SNAT4) can be selectively activated by an enzyme target (e.g. caspase-3/7), which triggers macrocyclization and subsequent in situ self-assembly into nanoaggregates retained at the target site. The tetrazine-imaging tag conjugate labels TCO in the nanoaggregates to generate selective signal retention for imaging in vitro, in cells, and in mice. Owing to the decoupling of enzyme activation and imaging tag immobilization, TCO-C-SNAT4 can be repeatedly injected to generate and accumulate more TCO-nanoaggregates for click labeling.
酶活性的靶向前成像尚未见报道，可能是由于缺乏将注射的底物在第一步保留下来用于后续标记的机制。在此，我们报告了两种生物正交反应——芳族腈和氨基硫醇的缩合反应以及四嗪和反式环辛烯（TCO）之间的逆电子需求 Diels-Alder 反应——的使用，以开发一种用于蛋白酶活性的靶向前成像的新策略。底物探针（TCO-C-SNAT4）可以被酶靶标（例如 caspase-3/7）选择性激活，这引发大环化和随后的原位自组装成在靶部位保留的纳米聚集体。四嗪成像标记物缀合物标记纳米聚集体中的 TCO，以产生体外、细胞内和小鼠内成像的选择性信号保留。由于酶激活和成像标记物固定的解耦，TCO-C-SNAT4 可以重复注射以产生和积累更多的 TCO-纳米聚集体用于点击标记。
